Bond & Jones: Stable-isotopeForum analysis for seabird biologists 183 A PRACTICAL INTRODUCTION TO STABLE-ISOTOPE ANALYSIS FOR SEABIRD BIOLOGISTS: APPROACHES, CAUTIONS AND CAVEATS

ALEXANDER L. BOND & IAN L. JONES

Department of Biology, Memorial University of Newfoundland, St. John’s, Newfoundland and Labrador, A1B 3X9, Canada ([email protected])

Received 21 May 2008, accepted 15 March 2009

SUMMARY

BOND, A.L. & JONES, I.L. 2009. A practical introduction to stable-isotope analysis for seabird biologists: approaches, cautions and caveats. Marine Ornithology 37: 183–188.

Stable isotopes of carbon and nitrogen can provide valuable insight into seabird diet, but when interpreting results, seabird biologists need to recognize the many assumptions and caveats inherent in such analyses. Here, we summarize the most common limitations of stable-isotope analysis as applied to ecology (species-specific discrimination factors, within-system comparisons, prey sampling, changes in isotopic ratios over time and biological or physiological influences) in the context of seabird biology. Discrimination factors are species specific for both the consumer and the prey species, and yet these remain largely unquantified for seabirds. Absolute comparisons across systems are confounded by differences in the isotopic composition at the base of each food web, which ultimately determine consumer isotopic values. This understanding also applies to applications of stable isotopes to historical seabird diet reconstruction for which historical prey isotopic values are not available. Finally, species biology (e.g. foraging behaviour) and physiologic condition (e.g. level of nutritional stress) must be considered if isotopic values are to be interpreted accurately. Stable-isotope ecology is a powerful tool in seabird biology, but its usefulness is determined by the ability of scientists to interpret its results properly.

Key words: δ13C, δ15N, assumptions, diet reconstruction, mixing model, seabird, stable isotopes

INTRODUCTION Isotopic ratios are expressed as a parts-per-thousand difference in the ratio of the heavier (more rare) to the lighter (more common) Stable-isotope ratio analysis is now commonly used by seabird isotope (i.e. 13C to 12C), compared with the ratio found in an biologists to infer diet and trophic relationships, to gain insight international standard (Pee Dee Belemnite for carbon, atmospheric into the foraging ecology of species, and to inform population air for nitrogen) such that management (Inger & Bearhop 2008). First recognized in the mid- 1980s (Peterson & Fry 1987), the use of stable-isotope analysis in avian ecology became widespread only after a series of experiments , [1] and field studies in the early 1990s (Hobson & Clark 1992a, 1992b, where δX is either δ13C or δ15N, and R is either the ratio 13C/12C or 1993; Hobson et al. 1994). However, as early as 1997, concerns 15N/14N. were raised about untested assumptions of the properties of stable isotopes and a lack of controlled laboratory experiments (Gannes et The value of δ15N increases predictably with increasing trophic al. 1997). Since then, considerable advances have been made (Wolf level, because 14N is excreted preferentially in nitrogenous waste et al. 2009), although in a recent thorough review of seabird diet (Steele & Daniel 1978, Minagawa & Wada 1984, Kelly 2000). studies and methods (Barrett et al. 2007), stable-isotope analysis The carbon ratio also changes, but in smaller amounts, and only was the sole common method for which biases and drawbacks were at lower trophic levels (DeNiro & Epstein 1978, Rau et al. 1983, not discussed thoroughly. As a result, seabird biologists who wish Hobson & Welch 1992). Moreover, carbon exhibits a gradient, to use stable-isotope analysis face a daunting and often massive with inshore food sources being enriched in 13C as compared with task to navigate the conflicting papers and knowledge gaps in the offshore sources in the marine environment (Peterson & Fry 1987, scientific literature. Considerable gaps remain in our knowledge Kelly 2000). Carbon can therefore potentially act as a geographic of how elemental isotopes behave in biological systems, and little identifier (Quillfeldt et al. 2005). controlled experimentation has been conducted. Here, we present an introduction to stable-isotope analysis for seabird biologists new to Isotopic ratios are determined at the time of tissue synthesis in the this emerging, yet widespread, tool. For brevity, we discuss only the consumer (Hobson & Clark 1992a) and therefore offer themselves isotopes commonly used in seabird studies: carbon and nitrogen. to non-destructive sampling in live animals (i.e. blood, feathers,

Marine Ornithology 37: 183–188 (2009) 184 Bond & Jones: Stable-isotope analysis for seabird biologists claws). These ratios can provide insight into seabird biology away such as muscle, liver and egg yolks almost certainly require lipid from the breeding colony if the proper tissue (e.g. moulted feathers) correction (Kojadinovic et al. 2008). is sampled. Tissue preservation DISCUSSION For many field studies, especially those involving seabirds on Lipids remote islands, the issue of tissue-preservation effects is of paramount importance. Formalin and genetic buffers can alter Compared with carbohydrates, lipids have less 13C because stable-isotope ratios drastically (Hobson et al. 1997, Gloutney & of fractionation caused by the oxidation of pyruvate to acetyl Hobson 1998), and results were mixed when tissues were preserved coenzyme A during lipid synthesis (DeNiro & Epstein 1977). in ethanol (Kaehler & Pakhomov 2001, Barrow et al. 2008). For Nevertheless, some researchers have found significant effects of avian tissue, freezing is the preferred method, but freezing may not lipid content on δ13C; others have not (McConnaughey & McRoy always be practical in the field, and so air drying (especially for 1979, Hobson & Clark 1992b, Pinnegar & Polunin 1999). blood samples) using an oven or similar smokeless heat source is also feasible (Bugoni et al. 2008). For a comprehensive review of Traditionally, lipids were removed from lipid-heavy tissues (C:N preservation techniques for stable-isotope samples, we direct the > 4.0) chemically (e.g. Bligh & Dyer 1959) to reduce variation reader to Barrow et al. (2008). in the isotopic ratio, but chemical extraction can also affect δ15N values (Murry et al. 2006). Two recent reviews (Post et al. 2007, Discrimination factors Logan et al. 2008) compared mathematical modelling methods and chemical extraction techniques, and concluded that analysing As prey nutrients are incorporated into the consumer, the isotopic a subset of samples before and after chemical lipid extraction will ratio changes by a “discrimination factor” (also called a “fractionation allow researchers to develop unique mathematical lipid models that factor”). In general, this factor falls between 0‰ and 2‰ for δ13C, can be applied to the remainder of the data in a given study. and between 2‰ and 5‰ for δ15N (Peterson & Fry 1987, Kelly 2000), and evidence is increasing that these ratios are unique to Seabird tissues such as feathers and egg albumen do not require each tissue–consumer–prey combination (Bearhop et al. 2002, lipid extraction (Kojadinovic et al. 2008), and blood typically does Cherel et al. 2005b, Caut et al. 2009). In addition, discrimination not. However, some Procellariiformes may have lipid-rich blood factors have long been regarded as an important aspect of stable- that would require lipid correction (Bond et al. 2010). Tissues isotope ecology (Mizutani et al. 1992) and are often applied poorly TABLE 1 Published mean discrimination factors for carbon and nitrogen stable isotopic ratios in seabird tissues based on a lipid-free fish dieta Species Consumer Discrimination factor (‰) Reference tissue C N King Penguin Aptenodytes patagonicus Whole blood –0.81 +2.07 Cherel et al. 2005b Feathers +0.07 +3.49 Cherel et al. 2005b Humboldt Penguin Spheniscus humboldtib Feathers +2.9 +4.8 Mizutani et al. 1992 Rockhopper Penguin Eudyptes chrysocome Whole blood +0.02 +2.72 Cherel et al. 2005b Feathers +0.11 +4.4 Cherel et al. 2005b Great Cormorant Phalacrocorax carbob Feathers +3.8 +3.7 Mizutani et al. 1992 Great Skua Stercorarius skua Whole blood +1.1 +2.8 Bearhop et al. 2002 Feathers +2.1 +4.6 Bearhop et al. 2002 Ring-billed Gull Larus delawarensis Whole blood –0.3 +3.1 Hobson & Clark 1992b Liver –0.4 +2.7 Hobson & Clark 1992b Muscle +0.3 +1.4 Hobson & Clark 1992b Bone collagen +2.6 +3.1 Hobson & Clark 1992b Feathers +0.2 +3.0 Hobson & Clark 1992b Black-tailed Gull L. crassirostrisb Feathers +5.3 +3.6 Mizutani et al. 1992 Common Murre Uria aalge Feather +1.2 +3.6 Becker et al. 2007 Rhinoceros Auklet Cerorhinca monocerata Whole blood — +3.49 Sears et al. 2009 a No discrimination factors have been published for members of the Diomedeidae, Procellariidae, Pelecanoididae, Hydrobatidae, Phaethontidae, Pelecanidae, Fregatidae, Sulidae or Rhyncopidae, or for other diets. b Lipids not extracted from prey items. Lipids result in a lower δ13C value, and therefore can change discrimination factors significantly.

Marine Ornithology 37: 183–188 (2009) Bond & Jones: Stable-isotope analysis for seabird biologists 185

(Caut et al. 2009). A recent review by Caut et al. (2009) provided their isotope signatures reflect the diet during the period of growth a decision tree for approximating discrimination factors for avian (Hobson & Clark 1992a). Therefore, a diet comparison across tissues, but we urge caution when applying these generalizations species using isotopic ratios from feathers grown away from the to marine birds, because the estimates were generated using non- breeding colony is invalid because there is no certainty concerning marine birds. Indeed, Caut et al. (2009) caution that other factors, the similarity of the isotopic composition of the food web, especially such as physiology, may play an important role in determining when the species of interest show geographic segregation. For many discrimination factors and should not be ignored by researchers. seabird species, winter diet and distribution are poorly known or completely unknown (Gaston & Jones 1998, Brooke 2004, Gaston To quantify discrimination factors accurately, consumers must be 2004, Barrett et al. 2007), and so a valid assessment or comparison held on a controlled, isotopically constant diet covering the length of isotopic ratios is challenging. Comparison of similar tissue types of time required for complete turnover of the tissue of interest would alleviate some of the potentially confounding factors. (Hobson & Clark 1992b). Most commonly, blood or feathers are sampled from seabirds. Whole-blood isotopic values are typically Isotope mixing models representative of diet for the previous 12–15 days (Hobson & Clark 1993); feathers indicate the isotopic ratios at the time of If discrimination factors are known or can be approximated, prey growth (Hobson & Clark 1992a). Even when feathers and blood and consumer isotopic ratios can be used in a mathematical model are synthesized over the same time period, consistent differences in to estimate the proportion of each prey group in the consumer’s stable-isotope ratios are detectable, with feathers being enriched in diet (Phillips & Gregg 2001, 2003). These models function on the both 15N and 13C as compared with blood (Quillfeldt et al. 2008). assumption that a plot of δ13C and δ15N of the prey species will create Proper assessment of discrimination factors therefore requires a polygon (mixing space) within which the consumer’s isotopic individuals to be held for lengthy periods of time—in some cases, ratios [corrected for discrimination, (see “Discrimination factors,” for months or years (Hobson & Clark 1992a, Becker et al. 2007). earlier)] will fall (Phillips & Gregg 2001, 2003). Depending on the specific question, models such as Isoerror, Isosource or MixSIR Maintaining seabirds in captivity for lengthy periods can be difficult are appropriate (Phillips & Gregg 2001, 2003; Phillips et al. 2005; (e.g. Oehler et al. 2001). Consequently, few discrimination factors Moore & Semmens 2008). In these models, ranges are given for the have been published (summarized in Table 1). Discrimination factors possible contribution of each prey source to the consumer’s isotope are cited consistently as the weakest link in stable-isotope ecology signature, but these can be very wide (e.g. Urton & Hobson 2005, (Phillips & Koch 2002, Post 2002, Cherel et al. 2005b), but they are Major et al. 2007) and meaningful biological interpretation can be essential for inferences about diet composition (see “Isotope mixing challenging, although not impossible (Cherel et al. 2005b). models,” later in this paper). When controlled laboratory studies are not possible, it may be possible to estimate discrimination factors Models are as useful as the data that go into them, and thus when from field studies (e.g. Bearhop et al. 2002). It should also be noted approximations are used in applying discrimination factors, the that captive and wild individuals may differ physiologically, which resulting model inherits and magnifies the uncertainty. Small could alone alter stable-isotope ratios. Should researchers wish to changes in discrimination factors can not only change the estimates make use of captive individuals, we encourage collaboration with of the proportions of each prey species, but also may dictate zoos and research centres having existing captive birds. whether the consumer’s isotope signature actually falls into the mixing space (the polygon bounded by source isotopic ratios on a Comparisons among and within food webs δ13C–δ15N plot). Critical to these models are proper discrimination factors (Caut et al. 2008, 2009). Recently, Bayesian models have The major assumption in stable-isotope ecology is that the signatures been able to incorporate the uncertainty in discrimination factors of consumers reflect those of their prey species, which is largely true (Moore & Semmens 2008). (Post 2002). Seabirds are multi-taxa predators, consuming a wide variety of prey species in an almost infinite number of combinations. Tissue heterogeneity This variation presents a problem when attempting to estimate the proportion of each prey item in the consumer’s diet (see “Isotope Within-tissue heterogeneity has received some attention in non- mixing models,” later in this paper), because different combinations of marine birds, with δD (ratio of hydrogen–deuterium) being assessed prey species and proportions can result in the same isotope signature within feathers (Wassenaar & Hobson 2006; Smith et al. 2008, in the consumer. It is therefore possible for two seabirds exploiting 2009), but interest is also emerging in how within- and between- two different food webs in the same location to have identical stable- individual isotope heterogeneity both affect the conclusions drawn isotope signatures. Although that example is extreme, many seabird from stable-isotope ratios (Jardine & Cunjak 2005). Only recently diets overlap during the breeding season (Ashmole 1963, Diamond have mixing models accounted for this uncertainly (Moore & 1978, Bearhop et al. 2004), and so making accurate estimates of Semmens 2008). In studies on captive fish, the inherent variability diet composition is often desired. Knowledge about the isotopic in captive and wild individuals ranged from about 2% to 10% for composition in the food web of interest is therefore required (Post δ15N and up to 19% for δ13C (Barnes et al. 2008). To date, tissue 2002). Such knowledge can be obtained relatively easily at seabird heterogeneity in δ13C and δ15N from avian tissue has not yet been breeding colonies by collecting food samples (Barrett et al. 2007). examined, and such heterogeneity remains a significant gap in Often a combination of traditional gut-content analysis and stable- advances of laboratory methodology. isotope analysis can provide valuable insight (Cherel et al. 2007). Reconstructing historical diet For migratory species, problems also arise when comparing isotopic values of tissues grown in different locations or at different Barrett et al. (2007) suggested that stable-isotope analysis is an ideal times of year. For example, feathers are inert once fully grown, and method for the reconstruction of historical diet, but those authors did

Marine Ornithology 37: 183–188 (2009) 186 Bond & Jones: Stable-isotope analysis for seabird biologists not review the inherent biases of the approach. Recently, two studies Nutritive stress and fasting are of particular concern, because (Becker & Beissinger 2006, Norris et al. 2007) attempted to quantify many seabird species fast either during incubation or because of the historical diet of Marbled Murrelets Brachyramphus marmoratus, spatial segregation between foraging and breeding grounds. A clear a threatened alcid from the west coast of North America (Nelson understanding of the physiology of the species of interest is critical 1997), by sampling feathers from museum specimens for stable to proper biologic interpretation of stable-isotope results. carbon and nitrogen isotopes. Based on changes in murrelet δ15N, it was estimated that Marbled Murrelets experienced a significant Foraging area may also change the isotopic composition in the decrease in trophic position and proportion of fish over the preceding tissues of consumers. For example, the Southern Ocean shows 100 years (Becker & Beissinger 2006, Norris et al. 2007). a latitudinal gradient in δ13C that is reflective of the isotopic composition of the base of the food web there (Quillfeldt et al. These studies lead us to a useful re-examination of some of 2005). Other areas remain untested; however, a gradient is likely the fundamental principles of stable-isotope ecology. Present-day present in all oceans (Goericke & Fry 1994). There is a great need isotopic ratios of prey items were used to infer historical diet, but to better understand the marine “isoscapes” of the world’s oceans it is impossible to know if the prey isotopic composition remained (Cherel et al. 2008). constant over time. Isotope signatures of Marbled Murrelets can change as drastically as 62% in trophic position (Norris et al. 2007), RECENT DEVELOPMENTS but prey diets (and consequently isotope signatures) may have changed as well (Quay et al. 2003). It may be that the proportion of Despite the heterogeneity and differences in stable-isotope ratios prey species changed, that the isotope signatures of prey changed or mentioned earlier, some researchers have been able to take that a combination of the two occurred. This situation is not testable, advantage of these differences to examine where species forage and it limits the valid inferences that can be made from historical data (e.g. Cherel et al. 2008) and consequently to better understand without detailed quantitative historical data from low trophic levels. at-sea mortality of seabirds through fisheries bycatch (Gómez-Díaz & González-Solís 2007). With continued research, and a growing Even when historical prey samples are available, isotopic ratios community of researchers using stable-isotope analysis, many of are prone to artificial changes caused by preservation techniques the potential pitfalls mentioned above will likely be overcome. (Hobson et al. 1997, Kaehler & Pakhomov 2001, Sarakinos et al. 2002, Feuchtmayr & Grey 2003, Rau et al. 2003). In addition, ACKNOWLEDGEMENTS some “baseline” δ13C values—those that ultimately determine the ratios in consumers (Post 2002)—may not remain constant over We thank R. Cunjak, K. Hobson and T. Jardine for valuable time, because burning of fossil fuel emits gases depleted in δ13C discussions on isotope ecology, and Y. Cherel and two anonymous as compared with background levels [dubbed the “Suess effect” reviewers for providing valuable comments on previous drafts of

(Keeling 1979, Quay et al. 2003)]. Changes in CO2, which is this manuscript. increasing in seawater over time (Louanchi & Hoppema 2000), may also affect δ13C values. When examined in southern waters, the REFERENCES change in δ13C was on the order of a decrease of between 0.009‰ and 0.018‰ per year (Hilton et al. 2006). ASHMOLE, N.P. 1963. The regulation of numbers of tropical oceanic birds. Ibis 103b: 458–473. Confounding biologic factors BARNES, C., JENNINGS, S., POLUNIN, N.V.C. & LANCASTER, J.E. 2008. The importance of quantifying inherent variability when Finally, a consideration of the biology of the focal species is crucial interpreting stable isotope field data. Oecologia 155: 227–235. to interpreting lab-generated data (Cherel & Hobson 2007). Factors BARRETT, R.T., CAMPHUYSEN, K., ANKER-NILSSEN, T., other than diet may influence isotopic composition in seabird CHARDINE, J.W., FURNESS, R.W., GARTHE, S., HÜPPOP, tissues, including foraging area (Quillfeldt et al. 2005, Cherel & O., LEOPOLD, M.F., MONTEVECCHI, W.A. & VEIT, R.R. Hobson 2007), body condition (Hobson et al. 1993) and metabolic 2007. Diet studies of seabirds: a review and recommendations. rate (Kitaysky 1999). Under nutritive stress, nitrogen is metabolised ICES Journal of Marine Science 64: 1675–1691. when proteins replace lipids as an energy source, resulting in BARROW, L.M., BJORNDAL, K.A. & REICH, K.J. 2008. Effects of changes to δ15N in some tissues, such as blood (Hobson et al. 1993, preservation method on stable carbon and nitrogen isotope values. Cherel et al. 2005a, Williams et al. 2007). Currently, the level of Physiological and Biochemical Zoology 81: 688–693. nutritive stress required to affect δ15N is not known, but as with BEARHOP, S., WALDRON, S., VOTIER, S.C. & FURNESS, R.W. discrimination factors, it is likely to be species-specific. Stress 2002. Factors that influence assimilation rates and fractionation of level may be of special interest to seabird biologists who use stable nitrogen and carbon stable isotopes in avian blood and feathers. isotopes to document diet shifts over time: When the proportion Physiological and Biochemical Zoology 75: 451–458. of a high-quality prey source decreases over time, at what point is BEARHOP, S., ADAMS, C.E., WALDRON, S., FULLER, R.A. & δ15N affected? MACLEOD, H. 2004. Determining trophic niche width: a novel approach using stable isotope analysis. Journal of Animal Ecology Because of metabolic differences, it is difficult to use stable isotopes 73: 1007–1012. to compare adult and chick diets (Williams et al. 2007, Harding et BECKER, B.H. & BEISSINGER, S.R. 2006. Centennial decline in al. 2008). Chicks are almost certainly metabolizing nutrients at the trophic level of an endangered seabird after fisheries decline. rate different from that of adults, resulting in different integration Conservation Biology 20: 470–479. periods for the same tissue (Sears et al. 2009). In addition, there BECKER, B.H., NEWMAN, S.H., INGLIS, S. & BEISSINGER, S.R. may be potential carryover effects from maternal nutrients in eggs; 2007. Diet–feather stable isotope (δ15N and δ13C) fractionation in more study is therefore required. Common Murres and other seabirds. Condor 109: 451–456.

Marine Ornithology 37: 183–188 (2009) Bond & Jones: Stable-isotope analysis for seabird biologists 187

BLIGH, E.G. & DYER, W.J. 1959. A rapid method of total lipid GOERICKE, R. & FRY, B. 1994. Variations of marine plankton δ13C

extraction and purification. Canadian Journal of Biochemistry with latitude, temperature and dissolved CO2 in the world ocean. and Physiology 37: 911–917. Global Biogeochemical Cycles 8: 85–90. BOND, A.L., McCLELLAND, G.T.W., JONES, I.L., LAVERS, GÓMEZ-DÍAZ, E. & GONZÁLEZ-SOLÍS, J. 2007. Geographic J.L. & KYSER, T.K. 2010. Stable isotopes confirm community assignment of seabirds to their origin: combining morphologic, patterns in foraging among Hawaiian Procellariiformes. genetic and biochemical analyses. Ecological Applications 17: Waterbirds 33: 50-58. 1484–1498. BROOKE, M. 2004. Albatrosses and petrels across the world. HARDING, A.M.A., HOBSON, K.A., WALKUSZ, W., DMOCH, K., Oxford, UK: Oxford University Press. KARNOVSKY, N.J., VAN PELT, T.I. & LIFJELD, J.T. 2008. Can BUGONI, L., McGILL, R.A.R. & FURNESS, R.W. 2008. Effects of stable isotope (δ13C and δ15N) measurements of Little Auk (Alle preservation methods on stable isotope signatures in bird tissues. alle) adults and chicks be used to track changes in high-Arctic Rapid Communications in Mass Spectrometry 22: 2457–2462. marine foodwebs? Polar Biology 31: 725–733. CAUT, S., ANGULO, E. & COURCHAMP, F. 2008. Caution on HILTON, G.M., THOMPSON, D.R., SAGAR, P.M., CUTHBERT, isotope model use for analyses of consumer diet. Canadian R.J., CHEREL, Y. & BURY, S.J. 2006. A stable isotopic Journal of Zoology 86: 438–445. investigation into the causes of decline in a sub-Antarctic predator, CAUT, S., ANGULO, E. & COURCHAMP, F. 2009. Variation the Rockhopper Penguin Eudyptes chrysocome. Global Change in discrimination factors (Δ15N and Δ13C): the effect of diet Biology 12: 611–625. isotopic values and applications for diet reconstruction. Journal HOBSON, K.A. & CLARK, R.G. 1992a. Assessing avian diets using of Applied Ecology 46: 443–453. stable isotopes I: turnover of 13C in tissues. Condor 94: 181–188. CHEREL, Y. & HOBSON, K.A. 2007. Geographical variation in HOBSON, K.A. & CLARK, R.G. 1992b. Assessing avian diets using carbon stable isotope signatures of marine predators: a tool to stable isotopes II: factors affecting diet–tissue fractionation. Condor investigate their foraging areas in the Southern Ocean. Marine 94: 189–197. Ecology Progress Series 329: 281–287. HOBSON, K.A. & CLARK, R.G. 1993. Turnover of 13C in cellular CHEREL, Y., HOBSON, K.A., BAILLEUL, F. & GROSCOLAS, and plasma fractions of blood: implications for nondestructive R. 2005a. Nutrition, physiology, and stable isotopes: new sampling in avian dietary studies. Auk 110: 638–641. information from fasting and molting penguins. Ecology 86: HOBSON, K.A. & WELCH, H.E. 1992. Determination of trophic 2881–2888. relationships within a high Arctic marine food web using δ13C and CHEREL, Y., HOBSON, K.A. & HASSANI, S. 2005b. Isotopic δ15N analysis. Marine Ecology Progress Series 84: 9–18. discrimination between food and blood and feathers of HOBSON, K.A., ALISAUSKAS, R.T. & CLARK, R.G. 1993. Stable- captive penguins: implications for dietary studies in the wild. nitrogen isotope enrichment in avian tissues due to fasting and Physiological and Biochemical Zoology 78: 106–115. nutritional stress: implications for isotopic analyses of diet. Condor CHEREL, Y., HOBSON, K.A., GUINET, C. & VANPE, C. 2007. 95: 388–394. Stable isotopes document seasonal changes in trophic niches and HOBSON, K.A., PIATT, J.F. & PITOCCHELLI, J. 1994. Using stable winter foraging individual specialization in diving predators from isotopes to determine seabird trophic relationships. Journal of the Southern Ocean. Journal of Animal Ecology 76: 826–836. Animal Ecology 63: 786–798. CHEREL, Y., LE CORRE, M., JAQUEMET, S., MÉNARD, HOBSON, K.A., GIBBS, H.L. & GLOUTNEY, M.L. 1997. F., RICHARD, P. & WEIMERSKIRCH, H. 2008. Resource Preservation of blood and tissue samples for stable-carbon and partitioning with a tropical seabird community: new information stable-nitrogen isotope analysis. Canadian Journal of Zoology 75: from stable isotopes. Marine Ecology Progress Series 366: 1720–1723. 281–291. INGER, R. & BEARHOP, S. 2008. Applications of stable isotope DENIRO, M.J. & EPSTEIN, S. 1977. Mechanism of carbon isotope analysis to avian ecology. Ibis 150: 447–461. fractionation associated with lipid synthesis. Science 197: JARDINE, T.D. & CUNJAK, R.A. 2005. Analytical error in stable 261–263. isotope ecology. Oecologia 144: 528–533. DENIRO, M.J. & EPSTEIN, S. 1978. Influence of diet on the KAEHLER, S. & PAKHOMOV, E.A. 2001. Effects of storage and distribution of carbon isotopes in animals. Geochimica et preservation on the δ13C and δ15N signatures of selected marine Cosmochimica Acta 42: 495–506. organisms. Marine Ecology Progress Series 219: 299–304. DIAMOND, A.W. 1978. Feeding strategies and population size in KEELING, C.D. 1979. The Suess effect: 13carbon and 14carbon tropical seabirds. American Naturalist 112: 215–223. interrelations. Environmental International 2: 229–300. FEUCHTMAYR, H. & GREY, J. 2003. Effect of preparation and KELLY, J.F. 2000. Stable isotopes of carbon and nitrogen in the study preservation procedures on carbon and nitrogen stable isotope of avian and mammalian trophic ecology. Canadian Journal of determinations from zooplankton. Rapid Communications in Zoology 78: 1–27. Mass Spectrometry 17: 2605–2610. KITAYSKY, A.S. 1999. Metabolic and developmental responses of GANNES, L.Z., O’BRIEN, D.M. & MARTINEZ DEL RIO, C. alcid chicks to experimental variation in food intake. Physiological 1997. Stable isotopes in animal ecology: assumptions, caveats and and Biochemical Zoology 72: 462–473. a call for more laboratory experiments. Ecology 78: 1271–1276. KOJADINOVIC, J., RICHARD, P., LE CORRE, M., COSSON, R.P. GASTON, A.J. 2004. Seabirds: a natural history. New Haven, CT: & BUSTAMANTE, P. 2008. Effects of lipid extraction on δ13C Yale University Press. and δ15N values in seabird muscle, liver and feathers. Waterbirds GASTON, A.J. & JONES, I.L. 1998 The auks: Alcidae. Oxford, 31: 169–178. UK: Oxford University Press. LOGAN, J.M., JARDINE, T.D., MILLER, T.J., BUNN, S.E., CUNJAK, GLOUTNEY, M.L. & HOBSON, K.A. 1998. Field preservation R.A. & LUTCAVAGE, M.E. 2008. Lipid corrections in carbon and techniques for the analysis of stable-carbon and nitrogen isotope nitrogen stable isotope analyses: comparison of chemical extraction ratios in eggs. Journal of Field Ornithology 69: 223–227. and modelling methods. Journal of Animal Ecology 77: 838–846.

Marine Ornithology 37: 183–188 (2009) 188 Bond & Jones: Stable-isotope analysis for seabird biologists

LOUANCHI, F. & HOPPEMA, M. 2000. Interannual variations POST, D.M., LAYMAN, C.A., ARRINGTON, D.A., TAKIMOTO,

of the Antarctic Ocean CO2 uptake from 1986–1994. Marine G., QUATTROCHI, J. & MONTAÑA, C.G. 2007. Getting to the Chemistry 72: 103–114. fat of the matter: models, methods and assumptions for dealing MAJOR, H.L., JONES, I.L., CHARETTE, M.R. & DIAMOND, with lipids in stable isotope analysis. Oecologia 152: 179–189. A.W. 2007. Variations in the diet of introduced Norway Rats QUAY, P., SONNERUT, R., WESTBY, T., STUTSMAN, J. & (Rattus norvegicus) inferred using stable isotope analysis. McNICHOL, A. 2003. Changes in the 13C/12C of dissolved inorganic

Journal of Zoology (London) 271: 463–468. carbon in the ocean as a tracer of anthropogenic CO2 uptake. Global McCONNAUGHEY, T. & McROY, C.P. 1979. Food-web structure Biogeochemical Cycles 17: 1004, doi:10.1029/2001GB001817. and the fractionation of carbon isotopes in the Bering Sea. QUILLFELDT, P., McGILL, R.A.R. & FURNESS, R.W. 2005. Diet Marine Biology (Berlin) 53: 257–262. and foraging areas of Southern Ocean seabirds and their prey MINAGAWA, M. & WADA, E. 1984. Stepwise enrichment of 15N inferred by stable isotopes: review and case study of Wilson’s along food chains: further evidence and the relation between Storm-Petrel. Marine Ecology Progress Series 295: 295–304. δ15N and animal age. Geochimica et Cosmochimica Acta 48: QUILLFELDT, P., BUGONI, L., McGILL, R.A.R., MASELLO, 1135–1140. J.F. & FURNESS, R.W. 2008. Differences in stable isotopes in MIZUTANI, H., FUKUDA, M. & KABAYA, Y. 1992. δ13C and blood and feathers of seabirds are consistent across species, age δ15N enrichment factors of feathers of 11 species of adult birds. and latitude: implications for food web studies. Marine Biology Ecology 73: 1391–1395. 155: 593–598. MOORE, J.W. & SEMMENS, B.X. 2008. Incorporating uncertainty RAU, G.H., MEARNS, A.J., YOUNG, D.R., OLSON, R.J., and prior information into stable isotope mixing models. Ecology SCHAFER, H.A. & KAPLAN, I.R. 1983. Animal 13C/12C Letters 11: 470–480. correlates with trophic level in pelagic food webs. Ecology 64: MURRY, B.A., FARRELL, J.M., TEECE, M.A. & SMYNTEK, 1314–1318. P.M. 2006. Effect of lipid extraction on the interpretation of fish RAU, G.H., OHMAN, M.D. & PIERROT-BULTS, A. 2003. Linking community trophic relationships determined by stable carbon nitrogen dynamics to climate variability off central California: and nitrogen isotopes. Canadian Journal of Fisheries and a 51 year record based on 15N/14N in CalCOFI zooplankton. Aquatic Sciences 63: 2167–2172. Deep-Sea Research Part II Tropical Studies in Oceanography NELSON, S.K. 1997. Marbled Murrelet (Brachyramphus 40: 2431–2447. marmoratus). In: Poole, A. & Gill, F. (Eds). The birds of SARAKINOS, H.C., JOHNSON, M.L. & VANDER ZANDEN, North America. No. 276. Philadelphia, PA: The Birds of North M.J. 2002. A synthesis of tissue-preservation effects on carbon America, Inc. and nitrogen stable isotope signatures. Canadian Journal of NORRIS, D.R., ARCESE, P., PREIKSHOT, D., BERTRAM, Zoology 80: 381–387. D.F. & KYSER, T.K. 2007. Diet reconstruction and historic SEARS, J., HATCH, S.A. & O’BRIEN, D.M. 2009. Disentangling population dynamics in a threatened seabird. Journal of Applied effects of growth and nutritional stress on seabird stable isotope Ecology 44: 875–884. ratios. Oecologia 159: 41–48. OEHLER, D.A., SCHMID, S.C. & MILLER, M.P. 2001. Maintaining SMITH, A.D., DONOHUE, K. & DUFFY, A.M. Jr. 2008. Intrafeather Least Auklet, Aethia pusilla, at the Cincinnati Zoo and Botanical and intraindividual variation in the stable-hydrogen isotope (δD) Garden: hand-rearing protocol with field, development and content of raptor feathers. Condor 110: 500–506. behavioural observations. Der Zoologische Garten (Neue Folge) SMITH, A.D., LOTT, C.A., SMITH, J.P., DONOHUE, K.C., 71: 316–334. WITTENBERG, S., SMITH, K.G. & GOODRICH, L. 2009. PETERSON, B.J. & FRY, B. 1987. Stable isotopes in ecosystem Deuterium measurements of raptor feathers: does a lack of studies. Annual Review of Ecology and Systematics 18: 293– reproducibility compromise geographic assignment? Auk 126: 320. 41–46. PHILLIPS, D.L. & GREGG, J.W. 2001. Uncertainty in source STEELE, K.W. & DANIEL, R.M. 1978. Fractionation of nitrogen partitioning using stable isotopes. Oecologia 127: 171–179. isotopes by animals: a further complication to the use of PHILLIPS, D.L. & GREGG, J.W. 2003. Source partitioning using variations in the natural abundance of 15N for tracer studies. stable isotopes: coping with too many sources. Oecologia 136: Journal of Agricultural Science 90: 7–9. 261–269. URTON, E.J.M. & HOBSON, K.A. 2005. Intrapopulation variation PHILLIPS, D.L. & KOCH, P.L. 2002. Incorporating concentration in Gray Wolf isotope (δ15N and δ13C) profiles: implications for dependence in stable isotope mixing models. Oecologia 130: the ecology of individuals. Oecologia 145: 317–326. 114–125. WASSENAAR, L.I. & HOBSON, K.A. 2006. Stable-hydrogen PHILLIPS, D.L., NEWSOME, S.D. & GREGG, J.W. 2005. isotope heterogeneity in keratinous materials: mass spectrometry Combining sources in stable isotope mixing models: alternative and migratory wildlife tissue subsampling strategies. Rapid methods. Oecologia 144: 520–527. Communications in Mass Spectrometry 20: 2505–2510. PINNEGAR, J.K. & POLUNIN, N.V.C. 1999. Differential WILLIAMS, C.T., BUCK, C.L., SEARS, J. & KITAYSKY, A.S. fractionation of δ13C and δ15N among fish tissues: implication 2007. Effects of nutritional restriction on nitrogen and carbon for the study of trophic interactions. Functional Ecology 13: stable isotopes in growing seabirds. Oecologia 153: 11–18. 225–231. WOLF, N., CARLETON, S.A. & MARTINEZ DEL RIO, C. 2009. POST, D.M. 2002. Using stable isotopes to estimate trophic position: Ten years of experimental animal isotopic ecology. Functional models, methods and assumptions. Ecology 83: 703–718. Ecology 23: 17–26.

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